Fig. 4: In vitro evaluation of engineered cell–cell communication through soluble ligands.

a, Schematic of in vitro activation experiment. Cocultured cancer cell lines secrete endogenous soluble factors that activate the cognate SNIPR, driving expression of the downstream BFP reporter. b, Left, in vitro activation of primary human T cells bearing TGFβ SNIPR in coculture with the respective cancer cell line (n = 3 technical replicates; mean ± s.d.). Right, TGFβ1 secretion of cancer cell lines, measured by ELISA (n = 2 technical replicates). Error bars represent s.d. c, Left, in vitro activation of primary human T cells bearing VEGF SNIPR in coculture with the respective cancer cell line (n = 3 technical replicates; mean ± s.d.). Right, VEGF secretion of cancer cell lines measured by ELISA (n = 2 technical replicates). d, In vitro activation of primary human T cells bearing VEGF SNIPR in transwell cell culture with the respective cancer cell line (n = 3 technical replicates; mean ± s.d.). e, Left, schematic of ligand blocking coculture experiment. BFP reporter activation of primary human TGFβ (middle) or VEGF (right) SNIPR T cells in coculture with A549 target cells, or with the addition of recombinant ligand. Activation was performed with and without a simultaneously administered blocking antibody and measured 24 h after treatment. Data are mean of n = 3 technical replicates ± s.d. Statistics computed using Welch’s unpaired two-tailed t-test. f, Relative killing efficacy of TGFβ SNIPR→CAR or constitutive HER2 CAR T cells targeting A549^RFP-NLS cells, measured by Incucyte live-cell imaging. hmSNIPR, hinge mutant SNIPR. g, Incucyte imaging of VEGF SNIPR→CAR or constitutive HER2 CAR T cells targeting A549^RFP-NLS cells.

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